Tobacco use is a leading contributor to disability and death worldwide. Smoking behaviors, including onset of smoking, smoking persistence (current smoking versus past smoking), and nicotine dependence, cluster in families, and large twin studies indicate that this clustering reflects genetic factors. Using over 2.4 million Single Nucleotide Polymorphisms (SNPs), we have performed a genome wide association (GWA) study using nicotine dependent smokers as cases and non-dependent smokers as controls. In addition, we targeted over 300 candidate genes for genotyping in the same series of cases and controls. Nicotine metabolites were measured in the blood of a subset of 196 smokers from our genetic study. Our GWA findings implicate several novel genes, including Neurexin 1, (NRXN1) in the development of nicotine dependence as well as identify a known candidate gene, the Ii3 nicotinic receptor. Significant associations were also observed in 23 candidate genes, including several nicotinic receptor (nAChR) genes. The goal of the current study is to follow up these findings to identify specific functional alleles that influence risk for nicotine dependence and to use in vivo measures of nicotine metabolism to identify functional alleles in nicotine metabolizing enzymes that may influence risk for dependence. We will achieve these goals by 1) Genotyping the most significant SNPs from each stage of our prior study in an independent case control series; 2) Performing fine mapping of genes that show evidence of replication and sequencing candidate genes, where necessary, to identify putative functional alleles; 3) Sequencing genes encoding the major nicotine metabolizing enzymes in individuals from the top and bottom tenth percentiles of the plasma cotinine, N-oxidation and N-glucuronidation distributions. Putative functional alleles in each gene will then be genotyped in all 196 individuals in whom nicotine metabolism was measured to determine whether the alleles are associated with differences in nicotine metabolism and, in the case control series, to determine whether they affect risk for nicotine dependence. Lastly, functional studies will be performed to determine the mechanism by which they affect risk for nicotine dependence. Initial studies will focus on the CHRNA5 gene because our preliminary data in two independent studies suggest that a non-synonymous coding change (D398N) results in changes in the sensitivity of the receptor that lead to changes in risk for nicotine dependence.